Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

Abstract

Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 37 degrees C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1(+) tumour cells with an IC(50) of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1(+) malignancies.

Figure 1

Schematic representation of fusion protein expression vector pBJR-2. Ap^R, ampicillin resistance gene; ColE1, origin of DNA replication; c-myc, sequence encoding the c-myc epitope; His₆, hexa-histidine encoding sequence; P/O, lac wild-type promoter/operator; rbs, ribosome-binding site; pelB, signal peptide sequence of bacterial pectate lyase; V_H, variable heavy chain; V_L, variable light chain; rapLR1, Rana pipiens liver ribonuclease 1; H71, framework 3 residue 71; G₄S, spacer connecting the ribonuclease with the scFv; (G₄S)₃, linker connecting the variable domains. Restriction sites for cloning are indicated.

Figure 2

Specific binding activity of mAb huHMFG1 and derived scFv fragments 4.10W and 4.9M. Flow cytometric analysis revealed for all purified antibodies binding to breast carcinoma cell line MCF7 and ovarian carcinoma cell line SKOV3, no binding was detected for T-cell lymphoma cell line Jurkat and mouse myeloma B-cell line SP2/0-AG14. Cells stained with secondary antibodies only were used as negative control.

Figure 3

Serum stability of scFv fragments. Immunoreactivity of wild-type scFvs 4.10W and 22W, and mutant variant scFvs 4.9M and 22M with MCF7 cells was measured by flow cytometry after incubating the constructs at 37°C in human serum for various time periods. Binding activity is shown as per cent of the maximal MFI at time point zero.

Figure 4

Effect of temperature on immunoreactivity and stability of mutant scFv 4.9M. (A) Binding was analysed by flow cytometry before and after incubation at 37°C for 1 h either in PBS or human serum. MCF7 cells stained with secondary antibodies were used as negative control. (B) Analytical size-exclusion FPLC on a calibrated Superdex 75 column was performed before (left panel) and after (right panel) incubation of 20 μg ml⁻¹ scFv in PBS at 37°C for 1 h. Monomeric protein eluted at 12.5 ml at a flow rate of 0.3 ml min⁻¹.

Figure 5

Homology modelling. The models shown as worm diagrams illustrate the influence of residue V_H-71 on the conformation of CDR-H1 and CDR-H2. Model-1 with the side chain of Arg71 exposed to the surface of the V_H domain is shown in red, Model-2 with Arg71 buried between motifs CDR-H1 and CDR-H2 in green, and Model-3 with Ala71 in blue.

Figure 6

Whole-cell ELISA of phage-displayed wild-type and mutant scFv. Various concentrations of 4.10 wild-type scFv phage, 4.9 mutant scFv phage, or control M13KO7 helper phage were incubated with MCF7 cells subconfluently grown in 96-well plates for 2 h at 37°C. Bound phages were detected with anti-M13-HRP polyclonal antibody as described in Materials and Methods.

Figure 7

Cytotoxicity of rapLR1-G₄S-4.9M fusion protein. Various concentrations of the fusion protein or RNase alone were incubated with MCF7 cells for 3 days and protein synthesis measured by [¹⁴C]leucine incorporation as described in Materials and Methods. Protein synthesis inhibition of target cells is expressed as percentage of protein synthesis to buffer-treated control cells.